LEVELS OF DNAK AND DNAJ PROVIDE TIGHT CONTROL OF HEAT-SHOCK GENE-EXPRESSION AND PROTEIN REPAIR IN ESCHERICHIA-COLI

The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the sigma 3 2 subunit of RNA polymerase, and negative modulators. Modulators are t he DnaK chaperone system, which inactivates and destabilizes sigma(32) , and the FtsH protease, which is largely responsible for sigma(32) de gradation. A yet unproven hypothesis is that the degree of sequestrati on of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dna J and ftsH from IPTG and arabinose-controlled promoters. Small increas es in levels of DnaK and the DnaJ co-chaperone (<1.5-fold of wild type ) resulted in decreased level and activity of sigma(32) at intermediat e temperature and faster shut-off of the heat shock response. Small de creases in their levels caused inverse effects and, furthermore, reduc ed the refolding efficiency of heat-denatured protein and growth at he at shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in el evated levels of heat shock proteins and a prolonged shutoff phase of the heat shock response. In contrast, a decrease in FtsH levels increa sed the sigma(32) levels, but the accumulated sigma(32) was inactive, indicating that sequestration of FtsH alone cannot induce the heat sho ck response efficiently. DnaK and DnaJ thus constitute the primary str ess-sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.